Cancer is characterized by abnormal regulation of cell growth, a process that ultimately depends on the correct expression and regulation of a large number of genes by transcription factors that can act as oncoproteins and tumor suppressors. In the previous granting period, we developed an experimental model involving a Src-inducible breast epithelial line (MCF-10A) that makes it possible to kinetically follow the process of cellular transformation, including the formation of cancer stem cells and mammospheres. Furthermore, we discovered an epigenetic switch between non-transformed and transformed cells mediated by a positive feedback loop that involves the inflammatory transcription factor NF-:B, Lin28 microRNA regulator, Let-7 microRNA, and interleukin 6. The central goal of this proposal is to elucidate the transcriptional regulatory circuits and underlying molecular mechanisms involved in the processes of cellular transformation and the formation of cancer stem cells. First, using molecular biological approaches, we will functionally dissect the regulatory pathway (i.e. the ordered series of steps involving transcription factors, direct target genes, microRNAs, and microRNA targets) by which an inflammatory signal leads to cellular transformation. We will address the relationship between the inflammatory response and transformation in the fibroblast model and other non-transformed cells lines as well as mechanistic analysis of promoters/enhancers of key target genes (e.g. Lin28 and IL6) in order to understand why and how the epigenetic switch occurs in MCF-10A, but not in other cells. In addition, having shown that miR-335 microRNA behaves similarly to Let-7 in many respects, we will perform similar mechanistic experiments to determine how miR-335 (and its targets tenascin C and Sox4 transcription factor) contributes to the process linking inflammation to cellular transformation. Second, we will obtain whole-genome profiles of mRNAs, microRNAs, transcription factor binding sites and chromatin features (via ChIP-sequencing), as well will genetically test transcription factors for their effects on gene expression genome-wide and on transformation. The resulting description will provide a whole-genome molecular view of the transformation process, identify direct targets of transcription factors relevant for transformation, provide critical information that will be followed up with detailed mechanistic experiments. Third, using the same logic and experimental approaches, we will identify genes, microRNAs, and regulatory pathways involved in the generation of cancer stem cells and the formation of mammospheres. Key genes and pathways implicated in these processes will be subjected to genetic and other mechanistic experiments to elucidate transcriptional circuitry. As an example, we will mechanistically pursue our recent finding that the miR-200 microRNA family is down-regulated in cancer stem cells and directly targets SUZ12, a component of the polycomb complex. Overall, this tightly integrated set of functional genomic and directed mechanistic experiments on isogenic models of cellular transformation will help elucidate transcriptional regulatory circuits involved in human cancer.